Answer to reviewers
Reviewer 1
Major compulsory revisions
1. In some experiments, the authors use serum deprivation to induce a more
contractile phenotype. Most often in the literature, this is done by supplementing
the medium with insulin/transferrin/selenium (ITS). Was this also done in the
present study? This is not clear to me. This is important, as in this case, cross-talk
between insulin and muscarinic receptors could also account for part of the effects.
Answer
As we describe in “materials and methods” the culture medium did not contain
insulin/transferrin/selenium (ITS).
2. In Figure 4, it is demonstrated that CCh at a concentration of 10 -9M already
produces effect. In fact, this effect is greater than the effect seen at higher CCh
concentrations. This is puzzling. The affinity of CCh is not that high, so how is this
explained?
Answer
After 24h of incubation of ASMC in the presence of CCh the mean [ 3H]thymidine
incorporation is (Mean±SEM, N=5) 2739±473, 2463±1177 and 1747±742 for the
concentrations of CCh 10-9M, 3x10-7M and 10-5M respectively. Statistical analysis
reveals no significant effect between these concentrations.
Similarly, after 72h incubation of ASMC in the presence of CCh the mean
[3H]thymidine incorporation is (Mean±SEM, N=5) 307±61, 102±45 and 271±121 for
the concentrations of CCh 10-9M, 3x10-7 M and 10-5M respectively. There is a
significant difference between the effect of 10-9M and 3x10-7 M CCh (P=0.03).
Although available data from literature reveal only the combined proliferative effect
of muscarinic agonists with growth factors, studies in fibroblasts demonstrate that
muscarinic agonists may have a mitogenic effect mainly via M3 muscarinic receptors
stimulations. Studies in other types of cells demonstrate that M2 stimulation has an
apoptotic effect. Our unpublished data demonstrate first that both M 2 antagonist
gallamine (30mM) and M2-M3 antagonist tiotropium (30nM) alone have a mitogenic
effect, probably by an allosteric modulation-stimulation of muscarinic receptors and
second in the presence of gallamine both ACh or CCh did not significantly affect
[3H]thymidine incorporation while tiotropium not only abolish the mitogenic effect
of ACh or CCh but also decrease significantly [3H]thymidine incorporation. Therefore
we can assume that M2 and M3 stimulation may have opposite effect on ASMC
proliferation. This opposite effect could explain the higher effect of ACh-CCh on
[3H]thymidine incorporation in lower doses.
control
10% FBS
+ACh 10-7 M
+CCh 10-9 M
3
[ H]thymidine incorporation (cpm)
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30000
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20000
7000
6000
5000
4000
3000
2000
1000
0
***
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** **
N=10
+gallamine 10mM
N=10
+tiotropium 30nM
N=10
In order to improve the manuscript we have removed Fig. 4. However, the increased
thymidine incorporation in cells incubated with ACh or CCh is displayed in Fig.7 of
the revised manuscript.
3. The manuscript suddenly takes an unexpected turn in Figure 7. Here, it is
demonstrated that prolonged serum deprivation is required to expose a mitogenic
effect of muscarinic receptor agonism, which is due to upregulation of the
muscarinic receptor. While these data are of interest, and in fact represent the key
novel data in the manuscript, in my opinion, these data should be presented directly
after Figure 4.
Answer
The order of the data has been changed and the above data, as well as data obtained
by cell counting are provided in Fig. 8 of the revised manuscript.
4. Further to point 3, these data call for additional experiments as the thymidine
data shown in Figures 4 and 6 are collected using an entirely different experimental
design and can therefore not be used in the context of the data shown in Figures 7
and further. My suggestion would be to remove Figure 4 and 6 from the manuscript
and present Figure 5 after the current Figure 8. If the authors wish to retain
thymidine incorporation data, these should be newly collected using the design
shown in Figure 7-8.
Answer
The data that are presented in the revised manuscript are according to your
suggestions. However, we maintained the thymidine experiments as they were
(Fig.7), since we believe that it is important to show that muscarinic receptors can
affect the capability of ASMCs to synthesize DNA even when they are not enriched
with muscarinic receptors (Discussion section page 16).
5. Can the reduced responsiveness to CCh and ACh in Figure 10-11 not simply be due
to desensitization/downregulation of the muscarinic receptor? Although I
understand the rationale for these experiments, this represents an alternative
explanation that at the very least should be discussed.
Answer
Data presented in this study leads us to the assumption that this effect is due to
downregulation of M3 receptors, since these receptors are the main receptors that
are involved in the cell contraction and their number is decreased in the cells that
have the proliferative phenotype. There might of course be other explanations for
the decrease in the cell responsiveness (Gosens et al 2004, reference 20).
Furthermore, we observed a decrease in smooth muscle protein expression after
prolonged incubation with muscarinic agonists. Since these proteins are necessary
for the cell contraction, their expression can affect the cell responsiveness to
contractile stimuli (Discussion section, page 15).
6. The paper by Gosens et al (2004) is not in agreement with the current findings
(discussion page 16) as this paper shows that the reduction in contractile protein
expression is not associated with increased proliferative behavior.
Answer
Even though Gosens et al 2004 found that in bovine tracheal smooth muscle
muscarinic agonists decrease the expression of contractile proteins, as well as the
cell responsiveness, which is in agreement with the results from our study, they did
not find any alteration in the proliferative capacity. However, our experiments were
performed in isolated rabbit tracheal smooth muscle cells and some differences can be due
to differences between animals or between tissue and isolated cells.
Minor compulsory revisions
1. Figure 1 and 2 can be merged into one Figure. In addition, please use arrows on
Figure 1 to clarify the phenotype that is meant in the text. Further, higher resolution
images and reduction of the background fluorescence for Figure 2 would improve
the quality of this Figure substantially.
Answer
We have added arrows showing the cells in question and the use of colored figures
rather than black and white has improved the quality of the figures. The data from
former Figure 2 are now presented in the new figures 2, 3 and 4 together with the
respective data from Western blot analysis of contractile protein expression.
2. The font used in Figure 3 and further is very difficult to read. Please provide a
better font quality.
Answer
We have changed the font.
3. It is unclear what the asterisks in Figure 3 indicate: significance relative to control
(same day) or control (day 0)?
Answer
The asterisks indicate significance relative to control of the same day. However, in
the revised manuscript we show the quantification in Western blots only from day
15, since the effect was more obvious that day (Fig. 2. 3. 4)
4. The Western blot in Figure 5A is of insufficient quality due to unequal loading.
Please provide a better example.
Answer
This figure was removed from the revised manuscript, since the information that
muscarinic agonists activate the MAPK and PI3K signaling pathways is provided by
the use of these pathways’ inhibitors in Fig.7 and Fig.9
5. PD89005 should be PD98059?
Answer
PD98059 is now corrected.
Reviewer 2
If the cell cycle of rabbit ASM cells is +/- 50 h, how do the authors explain:
1. the strong increase in cell number after 24 h of incubation with FBS and ACh/CCh?
2. the lack of further increase after 24 h when cells are stimulated for up to 5 days
with ACh/CCh, whereas FBS stimulation increases cell number up to day 4?
Answer
1. The cells that are incubated in the culture conditions represent a mixed
population of cells that have different phenotypes and therefore different
proliferative capabilities. We have observed increased proliferation in the first 24h of
incubation with other factors as well, such as insulin (Papagianni et al Exp Clin
Endocrinol Diabetes 2007,115(2):118–23) or gender hormones (Stamatiou et al
Steroids 2011, 76: 400–408).
2. FBS contains a mix of growth factors that are capable of sustaining the maximum
proliferation in ASMCs, whereas muscarinic agonists can stimulate the cell
proliferation through causing a shift of the cells to the proliferative phenotype, but
prolonged incubation can lead to a decrease in muscarinic receptors and therefore
the agonists can not affect the cell in the same degree.
The authors claim that the effect of ACh/CCh on DNA synthesis in dose-independent.
One could argue that this effect is dose-dependent (at 24 and 72 h) but that the
effect of these agonists decreases with increasing concentrations.
Answer
We do agree with your observation, as well as with the first reviewer’s 2 nd comment,
which is similar to yours. Our explanation is that after 24h of incubation of ASMC in
the presence of CCh the mean [3H]thymidine incorporation is (Mean±SEM, N=5)
2739±473, 2463±1177 and 1747±742 for the concentrations of CCh 10-9M, 3x10-7M
and 10-5M respectively. Statistical analysis reveals no significant effect between
these concentrations.
Similarly, after 72h incubation of ASMC in the presence of CCh the mean
[3H]thymidine incorporation is (Mean±SEM, N=5) 307±61, 102±45 and 271±121 for
the concentrations of CCh 10-9M, 3x10-7 M and 10-5M respectively. There is a
significant difference between the effect of 10-9M and 3x10-7 M CCh (P=0.03).
Although available data from literature reveal only the combined proliferative effect
of muscarinic agonists with growth factors, studies in fibroblasts demonstrate that
muscarinic agonists may have a mitogenic effect mainly via M3 muscarinic receptors
stimulations. Studies in other types of cells demonstrate that M2 stimulation has an
apoptotic effect. Our unpublished data demonstrate first that both M2 antagonist
gallamine (30mM) and M2-M3 antagonist tiotropium (30nM) alone have a mitogenic
effect, probably by an allosteric modulation-stimulation of muscarinic receptors and
second in the presence of gallamine both ACh or CCh did not significantly affect
[3H]thymidine incorporation while tiotropium not only abolish the mitogenic effect
of ACh or CCh but also decrease significantly [3H]thymidine incorporation. Therefore
we can assume that M2 and M3 stimulation may have opposite effect on ASMC
proliferation. This opposite effect could explain the higher effect of ACh-CCh on
[3H]thymidine incorporation in lower doses.
control
10% FBS
+ACh 10-7 M
+CCh 10-9 M
3
[ H]thymidine incorporation (cpm)
###
*
30000
**
###
20000
7000
6000
5000
4000
3000
2000
1000
0
***
*** **
** **
N=10
+gallamine 10mM
N=10
+tiotropium 30nM
N=10
In order to improve the manuscript we have removed Fig. 4. However, the increased
thymidine incorporation in cells incubated with ACh or CCh is displayed in Fig.7 of
the revised manuscript.
The authors hypothesize that prolonged serum-deprivation leads to the conditions
needed for the induction of proliferation by ACh/CCh. The DNA synthesis data the
authors show seem to contradict that as increased thymidine incorporation is
measured after 24 h stimulation. The cell number (MTT) however does only increase
after prolonged serum deprivation. It would be really helpful if the authors would
discuss this discrepancy in detail, so as to provide the readers with new insights and
potential explanations.
Answer
The data presented in the revised manuscript (Fig. 7, Fig.8 ) give a better explanation
of the observed effect. Muscarinic stimulation is capable of inducing increased
thymidine incorporation after 24h of serum starvation, but there are not enough
muscarinic receptors after this time of serum starvation in order to have an increase
in cell number as well. However, when the M3 expression was altered due to
prolonged starvation (7days) we did observe an increase in cell number as well, as
demonstrated by both MTT and Trypan blue cell counting (Fig. 8) (Discussion section
page 17).
The authors use both DNA synthesis and the MTT assay to evaluate proliferation.
These are both accepted methods which complement each other. However, in view
of the points raised above, I would suggest the authors resort to cell counting in
order to put these issues to rest. In addition, the advised protocol for proliferation
studies is 48-72 h serum-deprivation for arrest of cells followed by 24h (DNA
synthesis) or 48-96h (cell number) stimulation with growth factor. The current setup
is not optimal.
Answer
We have done cell counting experiments that are shown in Fig. 8 of the revised
manuscript. We have used the presented protocols for proliferation assessment in
the past and have found that in the cells we use they are the most suitable, since
48h allow us to have increased cpm count in DNA synthesis.
What is the explanation for the apparent discrepancy between the effects of the
signaling inhibitors in the DNA synthesis and the MTT assay?
Answer
Thymidine incorporation was assessed after 24h of serum starvation (Fig.7), while
cell number was estimated after 7days of serum starvation (Fig.8). The serum
deprivation is responsible for alterations in the cell phenotype (induction of the
contractile phenotype) that is accompanied by alteration in the receptor density and
expression on the cell surface. Therefore, there may be differences in the activation
of the signaling pathways that are involved in cell proliferation. However, both the
activation of PI3K and MAPK signaling pathways is necessary for ASMC proliferation
(Vignola et al 2003, reference 1).
The authors seem to suggest that the induction of a proliferative phenotype, induced
by ACh/CCh in their study goes hand in hand with decreased contractility/contractile
protein expression. The authors also use ref. 21 (Gosens, Eur J Pharmacol) in this line
of thought. Firstly, to point out the obvious, the authors are referring to the wrong
paper: Gosens Br J Pharmacol 2004 is the correct paper. Secondly, the Gosens study
clearly shows and thoroughly discusses the fact that the MCh-induced loss of
contractility and contractile protein expression is NOT related to an increase in
proliferative capacity, but rather molecular pathways involving calcium mobilization,
as prolonged KCl stimulation has the same effect which is inhibited by a calcium
channel inhibitor.
Answer
Gosens et al (Br J Pharmacol 2004, reference 20), have performed their experiments
on bovine tracheal smooth muscle. The differences between animals, since Gosens
uses bovine but we use rabbit trachea, as well as the fact that in that study the
smooth muscle proliferation was estimated in tissues, whereas our experiments
were performed in isolated primary cell cultures can be an explanation to the
observed differences. Even though Gosens et al show that the MCh effect on
proliferation might not be exclusive, since KCl stimulation had the same effect, in our
study we show that the downregulation in M3 receptors (Fig.5) goes hand in hand
with reduced responsiveness (Fig.6), as well as with the cell shift to the proliferative
phenotype (Fig. 1, 2, 3, 4, 7, 8).
The authors suggest that 7 days of serum-deprivation induces a contractile
phenotype. The data in this study actually suggests otherwise. Table 1 and Fig 3
show that these cells have not acquired a contractile phenotype as the % of
contractile protein-positive cells is not different between day 3 and 7, and the
contractile protein expression in cell lysates shows no difference in expression
between FBS and serum-free conditions on day 7. This somewhat, or perhaps largely,
undermines the hypothesis that the induction of a contractile phenotype is required
to enable ACh/CCh-induced proliferation.
Answer
In our experiments serum starvation for 7 days increased the percentage of cells that
express α-actin or SM-MHC. This result is not statistically significant. The effect of serum
starvation on the percentage of cells that express α-actin and SM-MHC is statistically
significant on day 15. The percentage of ASMCs expressed desmin remained relevant
constant after serum starvation for 3-15 days (see below table)
Days of culture of serum starved ASMCs
Percentage of
0
3
7
15
α-actin
56.6±3.0
16.3±4.0
19.5±2.3
41.2±6.0#
SM-MHC
34.4±13.5
12.7±0.8
18.5±2.6
19.9±1.4#
desmin
25.9±6.0
18.2±4.5
20.0±1.1
17.9±0.5
cells
No changes were measured in the ratio of α-actin, myosin heavy chain (MHC) or desmin to
β-actin in cells incubated for 3-15 days in serum free medium (table below).
Days of culture
Percentage of
cells
0
3
7
15
α-actin
0.57±0.13
0.66±0.11
0.42±0.99
0.89±0.10
SM-MHC
0.9±0.08
1.02±0.02
0.94±0.1
1.25±0.2
desmin
053±0.01
0.60±0.06
0.60±0.04
0.68±0.5
Even though, the above results did not demonstrate that serum deprivation for 7 days
causes a shift of ASMC phenotype to contractile, the purpose of our study was not the
investigation of the effect of serum deprivation on ASMC phenotype but the effect
muscarinic agonist. Our experiments clearly demonstrated the muscarinic agonist-induced
shift of ASMC phenotype to synthetic/proliferative one.
As far as the study of relevance of phenotype to ASMC proliferation is concerned, we
focused mainly on the results that clearly demonstrate that serum deprivation increased
significantly M3 receptors on ASMC, since [3H]NMS binding in serum deprived cells was
increase from 18.5±0.4 on day 3 to 27.5±0.9 and 35.03±8 on day 15 respectively (Fig. 5).
General point: the results should be discussed thoroughly; the discussion section is
too speculative and offers little explanation for the observed effects.
Answer
We have changed the Discussion section